Large-scale Conformational Dynamics Research Articles

NMR and Single-Molecule FRET Insights into Fast Protein Motions and Their Relation to Function.

Proteins often undergo large-scale conformational transitions, in which secondary and tertiary structure elements (loops, helices, and domains) change their structures or their positions with respect to each other. Simple considerations suggest that such dynamics should be relatively fast, but the functional cycles of many proteins are often relatively slow. Sophisticated experimental methods are starting to tackle this dichotomy and shed light on the contribution of large-scale conformational dynamics to protein function. In this review, we focus on the contribution of single-molecule Förster resonance energy transfer and nuclear magnetic resonance (NMR) spectroscopies to the study of conformational dynamics. We briefly describe the state of the art in each of these techniques and then point out their similarities and differences, as well as the relative strengths and weaknesses of each. Several case studies, in which the connection between fast conformational dynamics and slower function has been demonstrated, are then introduced and discussed. These examples include both enzymes and large protein machines, some of which have been studied by both NMR and fluorescence spectroscopies.

Local and Large-Scale Conformational Dynamics in Unfolded Proteins and IDPs. I. Effect of Solvent Viscosity and Macromolecular Crowding.

Protein/solvent interactions largely influence protein dynamics, particularly motions in unfolded and intrinsically disordered proteins (IDPs). Here, we apply triplet-triplet energy transfer (TTET) to investigate the coupling of internal protein motions to solvent motions by determining the effect of solvent viscosity (η) and macromolecular crowding on the rate constants of loop formation (kc) in several unfolded polypeptide chains including IDPs. The results show that the viscosity dependence of loop formation depends on amino acid sequence, loop length, and co-solute size. Below a critical size (rc), co-solutes exert a maximum effect, indicating that under these conditions microviscosity experienced by chain motions matches macroviscosity of the solvent. rc depends on chain stiffness and reflects the length scale of the chain motions, i.e., it is related to the persistence length. Above rc, the effect of solvent viscosity decreases with increasing co-solute size. For co-solutes typically used to mimic cellular environments, a scaling of kc ∝ η-0.1 is observed, suggesting that dynamics in unfolded proteins are only marginally modulated in cells. The effect of solvent viscosity on kc in the small co-solute limit (below rc) increases with increasing chain length and chain flexibility. Formation of long and very flexible loops exhibits a kc ∝ η-1 viscosity dependence, indicating full solvent coupling. Shorter and less flexible loops show weaker solvent coupling with values as low as kc ∝ η-0.75±0.02. Coupling of formation of short loops to solvent motions is very little affected by amino acid sequence, but solvent coupling of long-range loop formation is decreased by side chain sterics.

Local and Large-Scale Conformational Dynamics in Unfolded Proteins and IDPs. II. Effect of Temperature and Internal Friction.

Internal dynamics of proteins are essential for protein folding and function. Dynamics in unfolded proteins are of particular interest since they are the basis for many cellular processes like folding, misfolding, aggregation, and amyloid formation and also determine the properties of intrinsically disordered proteins (IDPs). It is still an open question of what governs motions in unfolded proteins and whether they encounter major energy barriers. Here we use triplet-triplet energy transfer (TTET) in unfolded homopolypeptide chains and IDPs to characterize the barriers for local and long-range loop formation. The results show that the formation of short loops encounters major energy barriers with activation energies (Ea) up to 18 kJ/mol (corrected for effects of temperature on water viscosity) with very little dependence on amino acid sequence. For poly(Gly-Ser) and polySer chains the barrier decreases with increasing loop size and reaches a limiting value of 4.6 ± 0.4 kJ/mol for long and flexible chains. This observation is in accordance with the concept of internal friction encountered by chain motions due to steric effects, which is high for local motions and decreases with increasing loop size. Comparison with the results from the viscosity dependence of loop formation shows a negative correlation between Ea and the sensitivity of the reaction to solvent viscosity (α) in accordance with the Grote-Hynes theory of memory friction. The Arrhenius pre-exponential factor (A) also decreases with increasing loop size, indicating increased entropic costs for loop formation. Long-range loop formation in the investigated sequences derived from IDPs shows increased Ea and A compared with poly(Gly-Ser) and polySer chains. This increase is exclusively due to steric effects that cause additional internal friction, whereas intramolecular hydrogen bonds, dispersion forces, and charge interactions do not affect the activation parameters.

Tuning the Baird Aromatic Triplet State Energy of Cyclooctatetraene to Maximize the Self-Healing Mechanism in Organic Fluorophores

Bright, photostable and non-toxic fluorescent contrast agents are critical for biological imaging. ‘Self-healing’ dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio both in the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at sub-millisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design (Pati et al., PNAS, 2020).

Detection of Single Molecules Using Stochastic Resonance of Bistable Oligomers.

Ultra-sensitive elements for nanoscale devices capable of detecting single molecules are in demand for many important applications. It is generally accepted that the inevitable stochastic disturbance of a sensing element by its surroundings will limit detection at the molecular level. However, a phenomenon exists (stochastic resonance) in which the environmental noise acts abnormally: it amplifies, rather than distorts, a weak signal. Stochastic resonance is inherent in non-linear bistable systems with criticality at which the bistability emerges. Our computer simulations have shown that the large-scale conformational dynamics of a short oligomeric fragment of thermosrespective polymer, poly-N-isopropylmethacrylamid, resemble the mechanical movement of nonlinear bistable systems. The oligomers we have studied demonstrate spontaneous vibrations and stochastic resonance activated by conventional thermal noise. We have observed reasonable shifts of the spontaneous vibrations and stochastic resonance modes when attaching an analyte molecule to the oligomer. Our simulations have shown that spontaneous vibrations and stochastic resonance of the bistable thermoresponsive oligomers are sensitive to both the analyte molecular mass and the binding affinity. All these effects indicate that the oligomers with mechanic-like bistability may be utilized as ultrasensitive operational units capable of detecting single molecules.

Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions

The functional cycle of many proteins involves large-scale motions of domains and subunits. The relation between conformational dynamics and the chemical steps of enzymes remains under debate. Here we show that in the presence of substrates, domain motions of an enzyme can take place on the microsecond time scale, yet exert influence on the much-slower chemical step. We study the domain closure reaction of the enzyme adenylate kinase from Escherichia coli while in action (i.e., under turnover conditions), using single-molecule FRET spectroscopy. We find that substrate binding increases dramatically domain closing and opening times, making them as short as ∼15 and ∼45 µs, respectively. These large-scale conformational dynamics are likely the fastest measured to date, and are ∼100-200 times faster than the enzymatic turnover rate. Some active-site mutants are shown to fully or partially prevent the substrate-induced increase in domain closure times, while at the same time they also reduce enzymatic activity, establishing a clear connection between the two phenomena, despite their disparate time scales. Based on these surprising observations, we propose a paradigm for the mode of action of enzymes, in which numerous cycles of conformational rearrangement are required to find a mutual orientation of substrates that is optimal for the chemical reaction.

Aminoglycoside interactions and impacts on the eukaryotic ribosome

Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6'-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.

Conformational Fluctuations as an Intrinsic Mechanism of Action: The Lipase-Specific Foldase of Pseudomonas Aeruginosa Studied by Hybrid Fluorescence Spectroscopy and MD Simulations

The description of protein function requires structural information, not only from static structural models but also of flexibility and dynamics. An investigation of highly flexible proteins or IDPs requires techniques sensitive to a variety of time- and distance-scales. Fluorescence (FRET, FCS) is known to provide good temporal and spatial coverage but it is a label-based technique and lacks atomistic-details. All-atom MD, on the other hand, is challenged by simulation of slow processes and accurate description of all interactions. The lipase-specific foldase (Lif) from Pseudomonas aeruginosa is a highly flexible protein with residual secondary and tertiary structure. It is mandatory to produce lipase A (LipA) in an enzymatically active conformation. Using a combination of fluorescence techniques (single molecule FRET, fluorescence lifetime analysis and fFCS) and all-atom MD we propose a description of structure and dynamics of Lif in relation to LipA. In the Lif:LipA complex, Lif forms flexible α-helical scaffold embracing LipA in headphone-like structure. In the unbound form, Lif does not stay in the hollow “headphone” conformation but rather exhibits large-scale conformational dynamics, where its α-helical structure undergoes reversible collapses and extensions as well as unfolding, on the sub-microsecond to sub-millisecond timescale. This process allows Lif to bind LipA despite the fickleness of structure. We show how the multitude of states in fast exchange provides a basis for binding. In addition we show how this hybrid approach benefits FRET (insight into the molecular structure) and MD (sampling).

Photon-by-Photon Hidden Markov Model Analysis for Microsecond Single-Molecule FRET Kinetics.

The function of biological macromolecules involves large-scale conformational dynamics spanning multiple time scales, from microseconds to seconds. Such conformational motions, which may involve whole domains or subunits of a protein, play a key role in allosteric regulation. There is an urgent need for experimental methods to probe the fastest of these motions. Single-molecule fluorescence experiments can in principle be used for observing such dynamics, but there is a lack of analysis methods that can extract the maximum amount of information from the data, down to the microsecond time scale. To address this issue, we introduce H2MM, a maximum likelihood estimation algorithm for photon-by-photon analysis of single-molecule fluorescence resonance energy transfer (FRET) experiments. H2MM is based on analytical estimators for model parameters, derived using the Baum-Welch algorithm. An efficient and effective method for the calculation of these estimators is introduced. H2MM is shown to accurately retrieve the reaction times from ∼1 s to ∼10 μs and even faster when applied to simulations of freely diffusing molecules. We further apply this algorithm to single-molecule FRET data collected from Holliday junction molecules and show that at low magnesium concentrations their kinetics are as fast as ∼104 s-1. The new algorithm is particularly suitable for experiments on freely diffusing individual molecules and is readily incorporated into existing analysis packages. It paves the way for the broad application of single-molecule fluorescence to study ultrafast functional dynamics of biomolecules.

Conformational Fluctuations as an Intrinsic Mechanism of Action of Lipase Foldase Studied by the High-Precision FRET Toolkit and MD Simulations

The lipase foldase (Lif) of P. aeruginosa is a steric chaperon, which acts as a conformational switch for the activation of lipase A (LipA). In the Lif:LipA complex, Lif forms flexible α-helical scaffold embracing LipA in headphone-like structure. In the unbound form, Lif does not stay in the hollow “headphone” conformation but rather exhibits large-scale conformational dynamics, where this α-helical structure undergoes reversible collapses and extensions on the microsecond to sub-microsecond timescale. This process allows Lif to bind LipA despite the fickleness of structure. Structural details of the process are studied by the fluorescence spectroscopic toolkit: a FRET distance landscape is investigated by ensemble lifetime-resolved FRET (eTCSPC), the timescales of dynamics by correlation techniques (filtered FCS) and overall FRET populations and dynamics by single-molecule MFD studies. The combination of these techniques allows drawing a kinetic scheme within the limiting states of the conformational ensemble of Lif, unraveling the mechanism of Lif action. The study is supported by all-atom MD simulations. The combination of accurate FRET, MFD, FRET positioning and screening (FPS) and fFCS with MD simulations in a hybrid approach provides in-depth insight in the complexity of the conformational dynamics of flexible proteins and protein complexes.

Wild-type and molten globular chorismate mutase achieve comparable catalytic rates using very different enthalpy/entropy compensations

The origin of the catalytic power of enzymes with a meta-stable native state, e.g. molten globular state, is an unsolved challenging issue in biochemistry. To help understand the possible differences between this special class of enzymes and the typical ones, we report here computer simulations of the catalysis of both the well-folded wild-type and the molten globular mutant of chorismate mutase. Using the ab initio quantum mechanical/molecular mechanical minimum free-energy path method, we determined the height of reaction barriers that are in good agreement with experimental measurements. Enzyme-substrate interactions were analyzed in detail to identify factors contributing to catalysis. Computed angular order parameters of backbone N-H bonds and side-chain methyl groups suggested site-specific, non-uniform rigidity changes of the enzymes during catalysis. The change of conformational entropy from the ground state to the transition state revealed distinctly contrasting entropy/enthalpy compensations in the dimeric wild-type enzyme and its molten globular monomeric variant. A unique catalytic strategy was suggested for enzymes that are natively molten globules: some may possess large conformational flexibility to provide strong electrostatic interactions to stabilize the transition state of the substrate and compensate for the entropy loss in the transition state. The equilibrium conformational dynamics in the reactant state were analyzed to quantify their contributions to the structural transitions enzymes needed to reach the transition states. The results suggest that large-scale conformational dynamics make important catalytic contributions to sampling conformational regions in favor of binding the transition state of substrate.

Capturing Large-Scale Conformational Dynamics of P-Glycoprotein by MD Simulations

P-glycoprotein is one of the most biomedically relevant ABC transporters, due to its frequent over-expression in cancer cells and involvement in their multidrug resistance. The crystal structure of murine P-glycoprotein has been reported in the nucleotide-free state. Employing molecular dynamics simulations we have investigated its conformation, dynamics and transport mechanism in a membrane environment. The simulation results show that, surprisingly, P-glycoprotein is capable of adopting an unusually wide range of conformations under the nucleotide-free conditions, highlighted by the degree of separation between the two nucleotide-binding domains (NBDs). During the equilibrium simulations, the distance between the two NBDs fluctuates over a range of at least 20 A, exhibiting both wider and narrower NBD openings than what is captured in the crystal structure. Moreover, we have been able to successfully dock ATP and Mg2+ in the Walker A motif, and generate an ATP-bound state that remains stable throughout the following 50 ns simulations. Interestingly, the presence of ATP and Mg2+ does not significantly alter the distribution of P-glycoprotein between open and closed conformations during the simulations, implying that the closed dimeric NBDs might be an innate conformation already existing in the nucleotide-free form, and the presence of nucleotide only stabilizes such a closed form. These results are in very close agreement, and in fact rationalize the reported high fluctuations at the NBDs measured in several other ABC exporters, which is possibly representing a fundamental difference in the transport mechanisms between ABC exporters and importers.

Gas-phase processes and measurements of macromolecular properties in solution: On the possibility of false positive and false negative signals of protein unfolding

Electrospray ionization mass spectrometry is increasingly applied to study protein behavior in solution, including characterization of their higher order structure, conformational dynamics and interactions with small ligands and other biopolymers. However, actual measurements of fundamental ionic parameters (mass and charge) take place in vacuum, and an array of gas phase processes occurring prior to protein ion detection and characterization may certainly affect them. While most previous studies were concerned primarily with the effect of gas phase processes on mass measurement (e.g., integrity of macromolecular complexes in the absence of solvent, non-specific interactions, etc.), the focus of our attention is the ionic charge. Charge state distributions of protein ions in ESI MS are often used to characterize large-scale dynamic processes in solution (such as protein unfolding). Formation of metastable protein aggregates either in the bulk of solution or in the electrosprayed droplets, their consequent transfer to the gas phase and asymmetric dissociation may give rise to a population of highly charged ions. Presence of such ions in ESI mass spectra usually indicates loss of native structure in solution. Therefore, studies of large-scale conformational dynamics in solution by monitoring protein ion charge state distributions should be carried out at low protein concentrations in order to minimize the occurrence of false positive signals of protein unfolding. The opposite phenomenon, absence of highly protonated ionic species in ESI spectra of unfolded proteins, does not occur even in the case of highly acidic proteins lacking a sufficient number of basic residues.

Insights into Allosteric Control of Vinculin Function from Its Large Scale Conformational Dynamics

Insights into Allosteric Control of Vinculin Function from Its Large Scale Conformational Dynamics

Large-Scale Conformational Dynamics of the HIV-1 Integrase Core Domain and Its Catalytic Loop Mutants

HIV-1 integrase is one of the three essential enzymes required for viral replication and has great potential as a novel target for anti-HIV drugs. Although tremendous efforts have been devoted to understanding this protein, the conformation of the catalytic core domain around the active site, particularly the catalytic loop overhanging the active site, is still not well characterized by experimental methods due to its high degree of flexibility. Recent studies have suggested that this conformational dynamics is directly correlated with enzymatic activity, but the details of this dynamics is not known. In this study, we conducted a series of extended-time molecular dynamics simulations and locally enhanced sampling simulations of the wild-type and three loop hinge mutants to investigate the conformational dynamics of the core domain. A combined total of >480 ns of simulation data was collected which allowed us to study the conformational changes that were not possible to observe in the previously reported short-time molecular dynamics simulations. Among the main findings are a major conformational change (>20 Å) in the catalytic loop, which revealed a gatinglike dynamics, and a transient intraloop structure, which provided a rationale for the mutational effects of several residues on the loop including Q 148, P 145, and Y 143. Further, clustering analyses have identified seven major conformational states of the wild-type catalytic loop. Their implications for catalytic function and ligand interaction are discussed. The findings reported here provide a detailed view of the active site conformational dynamics and should be useful for structure-based inhibitor design for integrase.

Studies of electron transport dynamics in photosynthetic reaction centers using fast temperature changes.

Rates of thermoinduced conformational transitions of reaction center (RC) complexes providing effective electron transport were studied in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria using methods of fast freezing and laser-induced temperature jump. Reactions of electron transfer from the primary to secondary quinone acceptors and from the multiheme cytochrome c subunit to photoactive bacteriochlorophyll dimer were used as probes of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. In contrast to the quinone complex, the thermoinduced transition of the macromolecular RC complex to the state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220-280 K accounts for tens of seconds. This transition is thought to be mediated by large-scale conformational dynamics of the macromolecular RC complex.

Thermodynamics and dynamics of histidine-binding protein, the water-soluble receptor of histidine permease. Implications for the transport of high and low affinity ligands

The bacterial histidine permease is a model system for ABC transporters (traffic ATPases). The water-soluble receptor of this permease, HisJ, binds l-histidine and l-arginine (tightly) and l-lysine and l-ornithine (less tightly) in the periplasm, interacts with the membrane-bound complex (HisQMP2) and induces its ATPase activity, which results in ligand translocation. HisJ is a two-domain protein; in the absence of ligand, the cleft between two domains is open and binding of substrate stabilizes the closed conformation. Surprisingly, various liganded HisJ forms display substantial differences in their physicochemical characteristics and capacity to induce the ATPase. This is due to either different effects of the individual ligands on the respective closed structures, or to different equilibria being reached for each ligand between the open liganded form and the closed liganded form [Wolf, A., Lee, K.C., Kirsch, J.F. & Ames, G.F.-L. (1996) J. Biol. Chem.271, 21243–21250]. In this work, time-resolved measurements of the decay of intrinsic HisJ fluorescence and of the decay of the anisotropy of the fluorescence, as well as the analysis of the steady-state near UV CD and fluorescence spectra, rule out the model in which the differences between liganded complexes reflect different equilibria. The decay of the anisotropy of the fluorescence shows that liganded complexes differ dramatically in their large-scale conformational dynamics. Differential scanning calorimetry (DSC) curves for the HisJ thermal unfolding are well described by a scheme of equilibrium two-state unfolding of two independent domains, which can be ascribed to the two-domain structure of HisJ. This is true both for apo-HisJ at various pH values, and for HisJ in the presence of its ligands at varying concentrations, at pH 8.3. The DSC and structural data suggest that all ligands interact more extensively with the larger domain. A qualitative model for the HisJ conformational dynamics employing the idea of a twisting movement of the domains is proposed, which explains the difference in the efficacy of the ATPase induction by the various liganded HisJ forms.

Thermodynamics and dynamics of histidine-binding protein, the water-soluble receptor of histidine permease. Implications for the transport of high and low affinity ligands.

The bacterial histidine permease is a model system for ABC transporters (traffic ATPases). The water-soluble receptor of this permease, HisJ, binds L-histidine and L-arginine (tightly) and L-lysine and L-ornithine (less tightly) in the periplasm, interacts with the membrane-bound complex (HisQMP2) and induces its ATPase activity, which results in ligand translocation. HisJ is a two-domain protein; in the absence of ligand, the cleft between two domains is open and binding of substrate stabilizes the closed conformation. Surprisingly, various liganded HisJ forms display substantial differences in their physicochemical characteristics and capacity to induce the ATPase. This is due to either different effects of the individual ligands on the respective closed structures, or to different equilibria being reached for each ligand between the open liganded form and the closed liganded form [Wolf, A. , Lee, K.C., Kirsch, J.F. & Ames, G.F.-L. (1996) J. Biol. Chem. 271, 21243-21250]. In this work, time-resolved measurements of the decay of intrinsic HisJ fluorescence and of the decay of the anisotropy of the fluorescence, as well as the analysis of the steady-state near UV CD and fluorescence spectra, rule out the model in which the differences between liganded complexes reflect different equilibria. The decay of the anisotropy of the fluorescence shows that liganded complexes differ dramatically in their large-scale conformational dynamics. Differential scanning calorimetry (DSC) curves for the HisJ thermal unfolding are well described by a scheme of equilibrium two-state unfolding of two independent domains, which can be ascribed to the two-domain structure of HisJ. This is true both for apo-HisJ at various pH values, and for HisJ in the presence of its ligands at varying concentrations, at pH 8.3. The DSC and structural data suggest that all ligands interact more extensively with the larger domain. A qualitative model for the HisJ conformational dynamics employing the idea of a twisting movement of the domains is proposed, which explains the difference in the efficacy of the ATPase induction by the various liganded HisJ forms.